Method of treating glycogen storage disease type III with human acid α-glucosidase

ABSTRACT

The present invention relates, in general, to Glycogen Storage Disease and, in particular, to a method of treating Glycogen Storage Disease-type-III and to compounds and compositions suitable for use in such a method.

This application is the U.S. national phase of International ApplicationNo. PCT/US2009/003993, filed 8 Jul. 2009, which designated the U.S. andclaims the benefit to U.S. Provisional Application No. 61/129,612, filedJul. 8, 2008, the entire contents of each of which are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates, in general, to Glycogen Storage Diseaseand, in particular, to a method of treating Glycogen StorageDisease-type-III and to compounds and compositions suitable for use insuch a method.

BACKGROUND

Glycogen debranching enzyme (GDE) is a multifunctional enzyme acting as1,4-α-D-glucan: 1,4-α-D-glucan 4-α-D-glycosyltransferase (E.C 2.4.1.25)and amylo-1,6-glucosidase (E.C 3.2.1.33) in glycogen degradation. Thetwo activities of the debranching enzyme are believed to reside atseparate sites on a single polypeptide chain with a molecular mass of174 kDa. The structure-function domain has not been studied in detail(Chen and Burchell, Glycogen storage disease. In: The Metabolic andMolecular Bases of Inherited Disease, C. R. Scriver et al, eds., 7^(th)Edition McGraw-Hill/New York, pp. 935-965 (1995); Bates et al, FEBSLett. 58:181-185 (1975); Gillard et al, Biochemistry 16:3978-3987(1977); Chapter 71 Kishnani P S, Koeberl D, Chen Y T. Glycogen StorageDiseases, in The Online Metabolic & Molecular Bases of InheritedDisease, Valle D, Beaudet A L, Vogelstein B, Kinzler K W, Antonarakis SE, Ballabio A, Scriver C R, Sly W S, Childs B, Editors (2008)). Thepredominant form of cDNA that encodes human debrancher has a 4596 bpcoding region and a 2371 bp 3′ nontranslated region (Yang et al, J.Biol. Chem. 267:9294-9299 (1992)). Tissue specific debrancher mRNAsexist. These isoforms differ at the 5′ nontranslated region and arebelieved to be generated by differential RNA transcription and splicingfrom a single debrancher gene (Bao et al, Gene 197:389-398 (1997)). Thehuman gene is localized to chromosome 1p21 (Yang-Feng et al, Genomics13:931-934 (1992)). The genomic structure of the human GDE gene has beendetermined and consists of 35 exons spanning ˜85 kb of DNA.

Debranching enzyme, together with phosphorylase, is responsible forcomplete degradation of glycogen. Liver and muscle are the two majororgans most active in glycogen metabolism. The primary function ofglycogen in these organs is different. In muscle, glycogen provides alocal fuel store for short-term energy consumption. In liver, itmaintains glucose homeostasis.

Genetic deficiency of glycogen debranching enzyme (Glycogen StorageDisease-type III, GSD-III) causes an incomplete glycogenolysis resultingin accumulation of glycogen with abnormally short outer chain in variousorgans. The commonly affected organs in GSD-III are liver, skeletalmuscle and heart. The disease is characterized by hepatomegaly,hypoglycemia, short stature, variable myopathy and cardiomyopathy.Patients with this disease vary remarkably, both clinically andenzymatically (Markowitz et al, Gastroenterology 105:1882-1885 (1993);Shen et al, J. Clin. Invest. 98:352-357 (1996); Telente et al, Annals.Intern. Med. 120:218-226 (1994)). Most patients have disease involvingboth liver and muscle (type IIIa), some patients (−15% of all GSD-IIIpatients) have only liver involvement (type IIIb), and, in rare cases,there is a selective loss of only one of the two GDE activities(glucosidase, (type IIIc) or transferase (type IIId)). Liver symptoms inGSD-III can improve with age and may disappear after puberty. Overtliver cirrhosis has been seen in some patients, some have developedhepatocellular carcinoma. Muscle weakness, though minimal duringchildhood, may become predominant in adults with onset in the third orfourth decade. These patients have slowly progressive proximal weaknessand distal muscle wasting and some patients become wheelchair bound.Even within the subgroup of patients who develop myopathy/cardiomyopathythere is clinical variability. Some patients have asymptomaticcardiomyopathy, some have early symptomatic cardiomyopathy leading todeath, and some have only muscle and no apparent heart involvement. Anabnormal electrocardiogram (ECG) with ventricular hypertrophy is afrequent finding and does not correlate with clinical severity. Normalserum creatine kinase levels do not rule out muscle enzyme deficiency.The biochemical subtypes do not predict clinical severity.

There appears to be no correlation between the amount of debrancherprotein and clinical severity (Yang et al, Am. J. Hum. Genet. 41:A28(1992)). To predict accurately at initial diagnosis whether myopathy orcardiomyopathy may occur, one must determine whether debranching enzymeactivity is deficient in muscle (Chen and Burchell, Glycogen storagedisease. In: The Metabolic and Molecular Bases of Inherited Disease, C.R. Scriver et al, eds., 7^(th) Edition McGraw-Hill/New York, pp. 935-965(1995)). It appears that muscle disease will not develop in patientswith GDE activity retained in muscle (Coleman et al, Annals of InternalMedicine 116:896-900 (1992)).

The variable phenotype is, in part, explained by differences intissue-specific expression of the defective enzyme. As pointed outabove, in type IIIc, enzyme is deficient in both liver and muscle, inIIIb there is enzyme deficiency only in liver. Unlike phosphorylase,which has tissue-specific isoenzymes encoded by different genes, at theprotein level and at the molecular level it appears that there are notissue-specific GDE isoenzymes in different tissues. Until now, it hasnot been understood in GSD-III how a single GDE gene, normally expressedin all tissues, can change expression in different tissues (Chen andBurchell, Glycogen storage disease. In: The Metabolic and MolecularBases of Inherited Disease, C. R. Scriver et al, eds., 7^(th) EditionMcGraw-Hill/New York, pp. 935-965 (1995)). Two mutations (17delAG andG6X), both located in exon 3 at amino acid codon 6, are exclusivelyfound in the GSD-IIIb (Shen et al, J. Clin. Invest. 98:352-357 (1996))suggesting that exon 3 is important in controlling tissue-specificexpression of the GDE gene.

Histology of the liver in these patients is characterized by a universaldistension of hepatocytes by glycogen and the presence of fibrous septa.Electron microscopy studies on muscle specimens have shown presence ofaccumulated glycogen beneath the sarcolemma and between myofibrils; theexcess glycogen not only disperses in the cytoplasm, but is also seen inthe lysosomes (Cornelio et al, Arch. Neurol. 41:1027-1032 (1984),Miranda et al, Ann. Neurol. 9:283-288 (1981)).

The detailed structural biology of GDE is not known, although severalfunctional domains of glycogen debranching enzyme have been proposedfrom enzymological studies and sequence comparison to other enzymes withsimilar catalytic function (Yang et al, J. Biol. Chem. 267:929409299(1992), Liu et al, Archives of Biochemistry and Biophysics 306:232-239(1993), Liu et al, Biochemistry 34:7056-7061 (1995), Jespersen et al,Journal of Protein Chemistry 12(6):791-805 (1993)). A region at theCOOH-terminal of the debranching enzyme could be a candidate forglycogen binding site (Yang et al, J. Biol. Chem. 267:9294-9299 (1992)),and 4 regions at N-terminal half of the enzyme bear sequence homology tothe catalytic sites identified or proposed in other amylolytic enzymes(Liu et al, Archives of Biochemistry and Biophysics 306:232-239 (1993),Jespersen et al, Journal of Protein Chemistry 12(6):791-805 (1993)).Aspartate at position 549 has been identified as the catalyticnucleophile in the transferase site of rabbit muscle glycogendebranching enzyme (Braun et al, Biochemistry 35:5458-5463 (1996)).

Currently there is no effective treatment for the disease. Hypoglycemiacan be controlled by frequent meals high in carbohydrates withcornstarch supplements or nocturnal gastric drip feedings. Patients withmyopathy have been given diets high in protein during the daytime plusovernight enteral infusion. In some patients, transient improvement insymptoms has been documented but there are no long-term datademonstrating that the high protein diet prevents or treats theprogressive myopathy (Chen and Burchell, Glycogen storage disease. In:The Metabolic and Molecular Bases of Inherited Disease, C. R. Scriver etal, eds., 7^(th) Edition McGraw-Hill/New York, pp. 935-965 (1995)). Theprogressive myopathy and/or cardiomyopathy is a major cause of morbidityin adults and patients with progressive liver cirrhosis and hepaticcarcinoma have been reported. While gene therapy delivery of a normal,functional gene into the diseased organ could ultimately cure thedisease, an ideal gene delivery vehicle that is reliable is currentlynot available. There is no living animal model for this disease. Dogsaffected with GSD-III have been reported (Gregory et al, J. Vet. Intern.Med. 21(1):40-46 (2007)), and a breeding colony is currently beingestablished.

Enzyme replacement therapy has been effective in diseases in which theresponsible enzymes/proteins exert their functions in extracellularfluids, such as adenosine deaminase deficiency, hemophilia, andα1-antitrypsin deficiency, or in a lysosomal location such as alysosomal storage disease. Enzyme replacement has not been explored indiseases in which the defective enzyme is present in cytosol (such asthe debranching enzyme in GSD-III), presumably due to the lack of anefficient and specific cellular uptake mechanism that delivers exogenousenzyme across the plasma membrane into the cytoplasm. Liposomes can fusewith plasma membrane and deliver their content, however, the use ofliposomes is compromised by lack of organ-specific tropism and clearanceby the reticulo-endothelial system (Mumtaz et al, Glycobiology1(5):505-510 (1991)). For an effective treatment for GSD-III, the enzymeshould be able to target muscle and heart as well as liver.

Cytoplasmic glycogen is normally digested by phosphorylase anddebranching enzyme; excess glycogen taken up by lysosomes throughautophagy can be digested by lysosomal acid α-glucosidase (GAA).Deficiency of debranching enzyme activity results in massiveaccumulation of glycogen having abnormally short outer branches. Theexcess glycogen in GSD-III resides not only in the cytoplasm but also inthe lysosomes (cytoplasm>lysosome) (Cornelio et al, Arch. Neurol.41:1027-1032 (1984), Miranda et al, Ann. Neurol. 9:283-288 (1981)). Thissuggests that the “normal” GAA activity in GSD-III may not be sufficientto digest all the excess glycogen. GAA is a lysosomal exo1,4-α-D-glucosidase that hydrolyzes both α-1,4 and α-1,6 linkages ofglycogen and can completely digest glycogen with and without abnormallyshort outer branches (Onodera et al, J. Biochem. 116:7-11 (1994)). GAAthus acts on glycogen with abnormally short outer branches such asaccumulates in GSD-III. As this glycogen in GSD-III accumulates both incytoplasm and lysosomes, providing more GAA may help to digest lysosomalglycogen and hence also cytoplasmic glycogen. It is postulated that, asthe lysosomes are cleared of glycogen, glycogen from the cytoplasmshuffles into them thus decreasing the total amount of accumulatedglycogen in GSD-III patients.

Deficiency of GAA causes Pompe disease (type II glycogen storagedisease), a fatal metabolic myopathy with accumulation of glycogen inlysosome and cytoplasm (lysosome>cytoplasm) (Hirschhorn, GlycogenStorage Disease Type II: Acid α-glucosidase (Acid Maltase) Deficiency.In: The Metabolic and Molecular Bases of Inherited Disease, C. R.Scriver et al, eds., 7^(th) Edition McGraw-Hill/New York, pp. 2443-2464(1995)). Enzyme replacement therapy with mannose-6-Phosphate(man-6-P)-rich precursor recombinant human GAA results in efficientman-6-P receptor mediated endocytosis of the enzyme followed byreduction of both lysosomal and cytoplasmic glycogen in fibroblasts (VanHove et al, Proc. Natl. Acad. Sci. 93:65-70 (1996)). In vivo, thisenzyme targets heart and muscle as well as liver and spleen followingintravenous injection in animals and in human Pompe patients. The rapidclearance of glycogen in Pompe fibroblasts when cultured in glucose-freemedium suggests a ready mobilization of glycogen from both lysosomal andcytoplasmic compartments (Van Hove et al, Proc. Natl. Acad. Sci.93:65-70 (1996), DiMauro et al, Pedatr. Res. 7:739-744 (1973)). It iscontemplated that cytoplasmic glycogen continuously shuffles throughlysosomes by autophagy for degradation.

The present invention results, at least in part, from the realizationthat administered GAA can reduce lysosomal glycogen in GSD-III patientsand ultimately also reduce cytoplasmic glycogen. Some of theadministered GAA may also go directly into the cytosol and reduce theglycogen. The invention provides a method of treating GSD-III (as wellas GSD-IV, -VI, -IX, XI and cardiac glycogenosis due to AMP-activatedprotein kinase gamma subunit 2 deficiency) based on the use of GAA.

SUMMARY OF THE INVENTION

The present invention relates generally to GSD. More specifically, theinvention relates to methods of treating GSD-III and to compounds andcompositions suitable for use in such methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The light microscopic appearance of the skeletal muscle biopsyfrom a GSD-IIIa patient. The purple (dark areas) staining glycogen ispresent as cytoplasmic lakes within myocytes. The glycogen is notmembrane bound within lysosomes but is free flowing within thecytoplasm.

FIG. 2. Cytoplasmic glycogen forming lakes within myocyte under EM in apatient with GSD-III.

FIG. 3. Lysosomal glycogen was seen within myocyte of a patient withGSD-III.

FIGS. 4A and 4B. Pattern of glycogen accumulation in muscle cellsderived from a normal subject (JA) and 3 GSD-III patients (MG, DK andBJ). Duplicate cultures were harvested at times indicated. Cellular GAAactivity (FIG. 4A) and glycogen content (FIG. 4B) were determined induplicates for each culture (mean±SD).

FIGS. 5A and 5B. Glycogen depletion in untreated muscle cells from anormal subject (JA) and 3 GSD-III patients (MG, DK and BJ) by glucosestarvation. GAA activity (FIG. 5A) and glycogen content (FIG. 5B) wereanalyzed in GSD-III muscle cells after 48-hour culturing (from Day 13 toDay 15) in differentiation medium with (w/) glucose or without (w/o)glucose. (*, p value<0.01; **, p<0.001).

FIGS. 6A and 6B. rhGAA uptake and glycogen reduction in GSD-III musclecells from patients MG, DK and BJ after 48-hour treatment (from Day 13to Day 15) with 100 μg of rhGAA. Muscle cells from normal subject (JA)were treated from Day 7 to Day 10. (*, p value<0.05; **, p<0.005; ***,p<0.001).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of treating GSD, particularlyGSD-III, by administering GAA to an individual suffering from thedisease. The invention also relates to the use of the enzyme, GAA, inthe manufacture of a medicament for the treatment of GSD (e.g.,GSD-III). As described herein, patients suffering from, for example,GSD-III can be treated by administering GAA on, for example, a regularbasis. Patients thus treated can be expected to demonstrate improvementof hypoglycemia, hepatomegaly, hepatic function, cardiac status, and/ormuscular strength, as well as a reduction of tissue glycogen levels.

The invention makes possible the treatment of GSD-III, includingGSD-type IIIa, type IIIb, type IIIc or type IIId. The invention alsomakes possible the treatment of other forms of GSD, including, but notlimited to, GSD-IV, -VI, -IX, XI and cardiac glycogenosis due toAMP-activated protein kinase gamma subunit 2 deficiency.

The terms, “treat” and “treatment,” as used herein, refer toamelioration of one or more symptoms associated with the disease,prevention or delay of the onset of one or more symptoms of the disease,and/or lessening of the severity or frequency of one or more symptoms ofthe disease. For example, treatment can refer to improvement ofhypoglycemia, growth retardation, hepatomegaly, and hepatic function(e.g., reduction of SGOT, SGPT); cardiac status (e.g., reduction,amelioration or prevention of the progressive cardiomyopathy, arrhythmiaand other cardiac manifestations that can be found, for example, inGSD-III), myopathy (e.g., exercise tolerance), reduction of glycogenlevels in tissue (e.g., liver and muscle) of the individual affected bythe disease, or any combination of these effects. Further, the treatmentmay prevent long term complications such as liver cirrhosis,hepatocellular carcinoma due to clearance of glycogen with an abnormalstructure. In one preferred embodiment, treatment includes improvementof liver symptoms, particularly, in reduction or prevention of GSD(e.g., GSD-III)-associated hypoglycemia, hepatomegaly and abnormal liverfunction. The terms, “improve,” “prevent” or “reduce,” as used herein,indicate values that are relative to a baseline measurement, such as ameasurement in the same individual prior to initiation of the treatmentdescribed herein, or a measurement in a control individual (or multiplecontrol individuals) in the absence of the treatment described herein. Acontrol individual is an individual afflicted with the same form of thedisease (e.g., GSD-III) as the individual being treated, who is aboutthe same age as the individual being treated (to ensure that the stagesof the disease in the treated individual and the control individual(s)are comparable).

The individual being treated can be an individual (infant, child,adolescent, or adult human) having GSD-III. The individual can haveresidual GDE activity, or no measurable activity. In another preferredembodiment, the individual is an individual who has been recentlydiagnosed with the disease. Early treatment (treatment commencing assoon as possible after diagnosis) is important to minimize the effectsof the disease and to maximize the benefits of treatment.

While the invention is described in detail with reference to GSD-III,the methods described herein can also be used to treat individualssuffering from other GSDs, including, but not limited to, GSD-IV, -VI,IX and -XI. The methods described herein can also be used in thetreatment of individuals suffering from cardiac glycogenosis due toAMP-activated protein kinase gamma subunit 2 deficiency.

In the methods of the invention, GAA (preferably, human GAA) isadministered to the individual. The GAA is in a form that, whenadministered, targets tissues such as the tissues affected by thedisease (e.g., liver, heart or muscle). In one preferred embodiment,human GAA is administered in its precursor form, as the precursorcontains motifs that allow efficient receptor-mediated uptake of GAA.Alternatively, a mature form of human GAA that has been modified tocontain motifs to allow efficient uptake of GAA into cells, can beadministered. In a particularly preferred embodiment, the GAA is theprecursor form of recombinant human GAA.

GAA is obtainable from a variety of sources. In a particularly preferredembodiment, recombinant human acid α-glucosidase (rhGAA) produced inChinese hamster ovary (CHO) cell cultures is used (see, e.g., Fuller, M.et al., Eur. J. Biochem. 234:903 909 (1995); Van Hove, J. L. K. et al.,Proc. Natl. Acad. Sci. USA 93:65 70 (1996) and U.S. Pat. No. 7,056,712).Production of GAA in CHO cells yields a product having glycosylationthat allows significant and efficient uptake of GAA in tissues such asheart and muscle. MYOZYME (alglucosidase alpha) Genzyme Corp.), or otherrecombinant human GAA, can be used in accordance with the invention.

The GAA has a specific enzyme activity in the range of about 1.0-8.0μmol/min/mg protein, preferably in the range of about 4.0-8.0μmol/min/mg protein. In one preferred embodiment, the GAA has a specificenzyme activity of at least about 1.0 μmol/min/mg protein; morepreferably, a specific enzyme activity of at least about 4.0 μmol/min/mgprotein; even more preferably, a specific enzyme activity of at leastabout 6.0 μmol/min/mg protein; and still more preferably, a specificenzyme activity of at least about 8.0 μmol/min/mg protein.

GAA can be administered alone, or in compositions or medicamentscomprising the GAA, as described herein. The compositions can beformulated with a physiologically acceptable carrier or excipient toprepare a pharmaceutical composition. The carrier and composition can besterile. The formulation should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, sugars such as mannitol, sucrose, or others,dextrose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc., as well as combinations thereof. The pharmaceuticalpreparations can, if desired, be mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring, flavoringand/or aromatic substances and the like which do not deleteriously reactwith the active compounds. In a preferred embodiment, a water-solublecarrier suitable for intravenous administration is used.

The composition or medicament, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan also be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose,magnesium carbonate, etc.

The composition or medicament can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in a preferred embodiment,a composition for intravenous administration typically is a solution insterile isotonic aqueous buffer. Where necessary, the composition canalso include a solubilizing agent and a local anesthetic to ease pain atthe site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water, saline or dextrose/water. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

The GAA can be formulated as neutral or salt forms. Pharmaceuticallyacceptable salts include those formed with free amino groups such asthose derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with free carboxyl groups such as thosederived from sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, etc.

GAA (or composition or medicament containing GAA) is administered by anappropriate route. In one embodiment, the GAA is administeredintravenously. In other embodiments, GAA is administered by directadministration to a target tissue, such as heart or muscle (e.g.,intramuscular). In yet another embodiment, GAA is administered orally.More than one route can be used concurrently, if desired.

GAA (or composition or medicament containing GAA) can be administeredalone, or in conjunction with other agents, such as antihistamines(e.g., diphenhydramine) or immunosuppressants or other immunotherapeuticagents which counteract anti-GAA antibodies. Possible immunomodulationstrategies include preventive tolerance induction either with initiationof therapy, or tolerance modulation after the development of inhibitoryantibodies. The term, “in conjunction with,” indicates that the agent isadministered at about the same time as the GAA (or compositioncontaining GAA). For example, the agent can be mixed into a compositioncontaining GAA, and thereby administered contemporaneously with the GAA;alternatively, the agent can be administered contemporaneously, withoutmixing (e.g., by “piggybacking” delivery of the agent on the intravenousline by which the GAA is also administered, or vice versa). In anotherexample, the agent can be administered separately (e.g., not admixed)but within a short time frame (e.g., within 24 hours) of administrationof the GAA. In one embodiment, GAA (or composition containing GAA) isadministered in conjunction with an immunosuppressive orimmunotherapeutic regimen designed to reduce amounts of, or preventproduction of, anti-GAA antibodies. For example, a protocol similar tothose used in hemophilia patients (Nilsson, I. M. et al., N. Engl. J.Med. 318:947 50 (1988)) can be used to reduce anti-GAA antibodies. In aparticularly preferred embodiment, the immunosuppressive orimmunotherapeutic regimen is begun prior to the first administration ofGAA, in order to minimize the possibility of production of anti-GAAantibodies. As an example, use of Rituximab, which eliminates mature Bcells expressing CD20, methotrexate, which acts on both B and T cells,or different combinations of such agents is possible (Mendehlson et al,N. Engl. J. Med. 360(2):194-195 (2009)).

GAA (or composition or medicament containing GAA) is administered in atherapeutically effective amount (i.e., a dosage amount that, whenadministered, for example, at regular intervals, is sufficient to treatthe disease, such as by ameliorating symptoms associated with thedisease, preventing or delaying the onset of the disease, and/or alsolessening the severity or frequency of symptoms of the disease, asdescribed above). The amount that will be therapeutically effective inthe treatment the disease will depend on the nature and extent of thedisease's effects, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays can optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed can also depend on the route of administration, and theseriousness of the disease, and should be decided according to thejudgment of a practitioner and each patient's circumstances. Effectivedoses can be extrapolated from dose-response curves derived from invitro or animal model test systems. In a preferred embodiment, thetherapeutically effective amount is less than about 40 mg enzyme/kg bodyweight of the individual, preferably in the range of about 1-40 mgenzyme/kg body weight, and even more preferably about 20 mg enzyme/kgbody weight or about 10 mg enzyme/kg body weight. The effective dose fora particular individual can be varied (e.g., increased or decreased)over time, depending on the needs of the individual. For example, intimes of physical illness or stress, or if anti-GAA antibodies becomepresent or increase, or if disease symptoms worsen, the amount can beincreased.

The therapeutically effective amount of GAA (or composition ormedicament containing GAA) can be administered at regular intervals,depending on the nature and extent of the disease's effects, and on anongoing basis. Administration at a “regular interval,” as used herein,indicates that the therapeutically effective amount is administeredperiodically (as distinguished from a one-time dose). The interval canbe determined by standard clinical techniques. In preferred embodiments,GAA is administered monthly, bimonthly, weekly, twice weekly, or daily.The administration interval for a single individual need not be a fixedinterval but can be varied over time, depending on the needs of theindividual. For example, in times of physical illness or stress, ifanti-GAA antibodies become present or increase, or if disease symptomsworsen, the interval between doses can be decreased.

In one preferred embodiment, a therapeutically effective amount of 20 mgenzyme/kg body weight is administered bi-monthly. In another preferredembodiment, a therapeutically effective amount of 10 mg enzyme/kg bodyweight is administered weekly or 5 mg enzyme/kg body weight isadministered twice weekly.

The invention additionally pertains to a pharmaceutical compositioncomprising human GAA, as described herein, in a container (e.g., a vial,bottle, bag for intravenous administration, syringe, etc.) with a labelcontaining instructions for administration of the composition fortreatment of GSD-III, such as by the methods described herein.

Certain aspects of the invention are described in greater detail in thenon-limiting Examples that follow. (See also U.S. Pat. No. 7,056,712.)

Example 1

GAA is a lysosomal exo 1,4-α-D-glucosidase that hydrolyzes both α-1,4and, α-1,6 linkage of glycogen. A highly efficient system has beendeveloped for producing human GAA which targets heart and muscle andcorrects glycogen accumulation in patients with Pompe disease. Theexcess glycogen in GSD-III resides not only in the cytoplasm but also inthe lysosome (Cornelio et al, Arch. Neurol. 41:1027-1032 (1984), Mirandaet al, Ann. Neurol. 9:283-288 (1981)). This suggests that excesscytoplasmic glycogen is shuffled more effectively into the lysosomesthan can be cleared by the “normal” GAA activity in GSD-III cells. It ishypothesized that cytoplasmic glycogen continuously shuffles throughlysosomes by autophagy for degradation, and that the administered GAAcan reduce lysosomal glycogen in GSD-III and ultimately also reducecytoplasmic glycogen.

Source of the Enzyme

High GAA producing CHO cells can be grown in expanded culture andrecombinant enzyme purified from the medium. Milligram quantities ofpurified GAA is available weekly as part of an ongoing project on thedevelopment of enzyme replacement therapy for Pompe disease. MYOZYME(Genzyme Corp.), or other rhGAA (preferably CHO-produced), is suitablefor use in the instant invention.

Tissue Source

Muscle samples can be obtained from patients with GSD-IIIa whose diseasehas been previously diagnosed by demonstrating debrancher activitydeficiency in both liver and muscle. Needle muscle biopsy from patientswith the diagnosis can be performed. It is expected that at least 3patients with a confirmed diagnosis of GSD-IIIa will be studied. Theneedle muscle biopsy can obtain 50-70 mg of tissues sufficient foranalysis. The procedure is less invasive than an open biopsy. Currently,skin fibroblasts from 9 GSD-IIIa patients are available. An IRB approvedprotocol is available that makes it possible to obtain muscle and skintissue from patients with GSD III.

Cell Culture

Cultured skin fibroblasts and muscle cells can be used to test thefeasibility of using GAA to treat GSD-III. The focus will beparticularly on IIIa patients who have, in addition to hepatomegaly,progressive myopathy (increasing muscle weakness by muscle strengthtesting in 6 months to 1 year follow-up and/or clinical complaints of adecrease in muscle strength) and cardiomyopathy (increase in leftventricular mass in a 6-12 month period on follow-up), and also patientswith progressive liver disease. Deficiency of debranching enzyme andaccumulation of glycogen are present in skin fibroblasts and musclecells grown in culture from GSD-III patients (Miranda et al, Ann.Neurol. 9:283-288 (1981), DiMauro et al, Pedatr. Res. 7:739-744 (1973),Yang et al, Am. J. Hum. Genet. 47:735-739 (1990), Brown, Diagnosis ofglycogen storage disease, In: Wapnir Pa., (ed)., Congenital MetabolicDisease Diagnosis and Treatment, Dekker, New York, pp. 227-250 (1985)).Cultured muscle cells also reflect muscle biopsy findings in that bothcytoplasmic and lysosomal glycogen are observed (Cornelio et al, Arch.Neurol. 41:1027-1032 (1984), Miranda et al, Ann. Neurol. 9:283-288(1981)). The skin fibroblasts and muscle cultures can, therefore, beused to improve understanding of the pathogenesis of the disease and toevaluate approaches to therapy. Muscle cultures can be established frommuscle biopsy samples using a protocol similar to the isolation of quailmyoblasts (Konigsberg, Methods in Enzymology 45:511-527 (1979)).Replating of the primary cultures can be performed to select againstfibroblasts. If the biopsy sample is too small, a more efficientmyoblasts isolation method using the fluorescence-activated cell sortercan be performed (Webster, Experimental Cell Research 174:252-265(1988)). Myoblasts can be allowed to differentiate to myotubes bychanging the medium to 2% horse serum. Muscle cells in the appropriatestage will be used for conduct of the experiments.

Effect on Glycogen with GAA Treatment

Glycogen concentrations in skin fibroblasts vary with cell confluenceand passage number (DiMauro et al, Pedatr. Res. 7:739-744 (1973)).Experiments can be performed at near confluence, and each patient canserve as his/her own control by concurrent duplicate testing with andwithout GAA treatment. Dose response (from 500 to 5000 nmol/hr/ml ofGAA) and time course (1 to 10 days) can be tested for the effect ofglycogen reduction. Glycogen content can be measured in total cellhomogenates and also individually in cytosol and crude lysosomalfractions (van der Ploeg et al, J. Neurol. Sci. 79:327-336 (1987)).Electromicroscopic examination can be performed for evidence ofclearance in both cytoplasm and lysosomes.

To avoid obscuring of the results by the continuous glycogen synthesisthat occurs in cells cultured in the presence of glucose in the medium,cells can be shifted to glucose-free medium 24 hours after GAAtreatment. Deprivation of glucose resulted in greater than 80% drop ofglycogen in the normal cells, but only 30% reduction in GSD-III cells(Yang et al, Am. J. Hum. Genet. 47:735-739 (1990), Brown, Diagnosis ofglycogen storage disease, In: Wapnir P A, (ed)., Congenital MetabolicDisease Diagnosis and Treatment, Dekker, New York, pp. 227-250 (1985)).Thus sufficiently high levels of glycogen persist which allow accurateevaluation of GAA treatment. The persistent glycogen in GSD-III cellshave short outer brancher which can be assessed usingglucose-1-phosphate formed from endogenous polysaccharide byphosphorylase (Yang et al, Am. J. Hum. Genet. 47:735-739 (1990)).

Example 2 below includes a description of studies that have beenundertaken.

Example 2

Primary human skeletal muscle cultures from GSD-IIIa patients have beenestablished as an in vitro model to evaluate efficacy of rhGAA fortreatment of GSD-III. Myoblasts were isolated from three GSD-IIIapatients and one healthy volunteer. Histopathology of these musclebiopsies was examined by light and electronic microscopy (EM) to confirmabnormal glycogen accumulation in these GSD-IIIa patients. The lightmicroscopic appearance of the skeletal muscle biopsies from GSD-IIIapatients showed abundant glycogen accumulation in cytoplasmic pools(FIG. 1). Under EM, the vast majority of the glycogen was found free inthe cytoplasm along with small amount of membrane-bound glycogen (FIGS.2 and 3).

Differentiation of myoblasts into mature myotubes (myogenesis) wasinduced by incubation of the muscle cells in low-serum differentiation

medium (low-glucose DMEM (GIBCO) containing 2% Hyclone H1 horse serum(Sigma), 0.5 mg/ml Fetuin, 0.5 mg/ml BSA, 0.025 mg/ml gentamycin and0.125 μg/ml Amphotericin B (Clonetics). Glycogen was stored in fullydifferentiated GSD-III muscle cells when sufficient glucose was suppliedin the medium (FIG. 4). Incomplete glycogenolysis was seen in GSD-IIIcells, but not in normal control cells after 48-hour glucose starvation(FIG. 5), indicating lack of debranching enzyme activity in the GSD-IIIpatients. Fully differentiated GSD-III myotubes were treated for 48hours by adding 100 μg of rhGAA (i.e., MYOZYME (Genzyme)) into theculture medium. GAA enzyme activity and glycogen content were analyzedbiochemically and histologically in these cells. Treatment with rhGAAsignificantly reduced glycogen level by 48%, 35% and 17%, respectively,in the three GSD-IIIa patient muscle cells (FIG. 6). These data suggestthe role of GAA in glycogen clearance in conditions where the primarydefect results in cytoplasmic glycogen accumulation.

What is claimed is:
 1. A method of treating glycogen storage diseasetype III comprising administering to a human in need of such treatmentan amount of human acid α-glucosidase at a regular interval sufficientto effect said treatment.
 2. The method of claim 1, wherein the amountof the human acid α-glucosidase administered at the regular interval isless than about 40 mg of acid α-glucosidase per kilogram of body weightof the individual.
 3. The method of claim 1, wherein the human acidα-glucosidase is recombinant human acid α-glucosidase or a precursor ofrecombinant human acid α-glucosidase.
 4. The method of claim 3, whereinthe recombinant human acid α-glucosidase is produced in Chinese hamsterovary cells.
 5. The method of claim 1, wherein the regular interval ismonthly, bimonthly, weekly, twice weekly or daily.
 6. The method ofclaim 1, wherein the human acid α-glucosidase is administeredintravenously.
 7. The method of claim 1, wherein the human acidα-glucosidase is administered in conjunction with, or subsequent to,administration of an immunosuppressant.
 8. The method of claim 1,wherein the human acid α-glucosidase is administered intramuscularly,intrathecally or intraventricularly.
 9. A method of treating anindividual who has been diagnosed as having glycogen storage diseasetype III comprising administering by injection to said individualfollowing diagnosis of glycogen storage disease type III, an amount ofhuman acid α-glucosidase at a regular interval sufficient to effect saidtreatment.